Trace fossils as mechanical discontinuities in shales, insight for the generation of bedding-parallel veins (BPV).

Understanding shale petrophysical parameters is of interest due to its direct implications as cap rocks for CO2 or hydrogen storage, waste depositions, and as unconventional reservoirs. The generation and propagation of natural and induced fracture networks in such rocks is highly dependent on the mechanical behavior linked to several sedimentological parameters, as lithological discontinuities or bioturbation. This study is focused on a different sedimentological parameter that consists of trace fossils and their implication on the generation of fluid-assisted fractures, called bedding-parallel veins. In the Austral-Magallanes Basin, Southern Patagonia, Argentina, both geological features, Skolithos Ichnofacies (doomed pioneers trace fossils) and bedding-parallel veins, are numerous, especially at the top of the turbiditic bodies. The trace fossils exhibit U-shaped vertically oriented burrows composed of clean sandstone, partially cemented by calcite, and a spreite in the central part with heterogenous laminated siltstone. Bedding-parallel veins are composed of calcite fibers with some pyrite grains and bitumen. They are located on the top of the trace fossils along the lithological discontinuity between the turbiditic bodies and the impermeable shales. On their surfaces, a radial pattern starts growing from the trace fossils. Moreover, the number of bedding-parallel veins is dependent on the bioturbation intensity. With this study, we infer that trace fossils represent ichnological mechanical discontinuities (IMD) that have a key role in the generation and development of bedding-parallel veins. By correlation, we also suggest that these geological features must be thoroughly studied, especially regarding their potential for the development of induced fracturing networks.

Eosinophils of patients with localized and diffuse cutaneous leishmaniasis: Differential response to Leishmania mexicana, with insights into mechanisms of damage inflicted upon the parasites by eosinophils.

Eosinophils are mainly associated with parasitic infections and allergic manifestations. They produce many biologically active substances that contribute to the destruction of pathogens through the degranulation of microbicidal components and inflammatory tissue effects. In leishmaniasis, eosinophils have been found within inflammatory infiltrate with protective immunity against the parasite. We analyzed the responses of eosinophils from patients with localized (LCL) and diffuse (DCL) cutaneous leishmaniasis, as well as from healthy subjects, when exposed to Leishmania mexicana. All DCL patients exhibited blood eosinophilia, along with elevated eosinophil counts in non-ulcerated nodules. In contrast, only LCL patients with prolonged disease progression showed eosinophils in their blood and cutaneous ulcers. Eosinophils from DCL patients secreted significantly higher levels of IL-6, IL-8, and IL-13, compared to eosinophils from LCL patients. Additionally, DCL patients displayed higher serum levels of anti-Leishmania IgG antibodies. We also demonstrated that eosinophils from both LCL and DCL patients responded to L. mexicana promastigotes with a robust oxidative burst, which was equally intense in both patient groups and significantly higher than in healthy subjects. Coincubation of eosinophils (from donors with eosinophilia) with L. mexicana promastigotes in vitro revealed various mechanisms of parasite damage associated with different patterns of granule exocytosis: 1) localized degranulation on the parasite surface, 2) the release of cytoplasmic membrane-bound "degranulation sacs" containing granules, 3) release of eosinophil extracellular traps containing DNA and granules with major basic protein. In conclusion, eosinophils damage L. mexicana parasites through the release of granules via diverse mechanisms. However, despite DCL patients having abundant eosinophils in their blood and tissues, their apparent inability to provide protection may be linked to the release of cytokines and chemokines that promote a Th2 immune response and disease progression in these patients.

Antimicrobial and anti-inflammatory activity of Cystatin C on human gingival fibroblast incubated with Porphyromonas gingivalis.

Background: Periodontal disease is considered one of the most prevalent chronic infectious diseases, often leading to the disruption of tooth-supporting tissues, including alveolar bone, causing tooth mobility and loss. Porphyromonas gingivalis is considered the major etiological agent of this disease, having a plethora of virulence factors, including, lipopolysaccharides (LPS), hemolysins, and proteinases. Antimicrobial peptides are one of the main components of the innate immune response that inhibit the growth of P. gingivalis. The aim of this study was to analyze the antimicrobial activity of cystatin C and to assess the effect on the inflammatory and anti-inflammatory cytokines, the production of reactive oxygen species, and in the release of nitric oxide by human gingival fibroblasts incubated with P. gingivalis in the presence and absence of cystatin C. Methods: P. gingivalis ATCC 33277 was exposed to cystatin C for 24h and co-cultured with human gingival fibroblasts (HGFs) ATCC CRL-2014. The effect of cystatin on growth of P. gingivalis and HGFs was evaluated. Pro-inflammatory (TNFα, IL-1ß) and anti-inflammatory (IL-10) cytokines were determined by ELISA in the supernatants of HGFs incubated with P. gingivalis exposed to cystatin C. Additionally, nitrites and reactive oxygen species (ROS) production were evaluated. Results: Cystatin Cinhibited the growth of P. gingivalis without affecting HGFs. Incubation of HGFs with P. gingivalis led to a significant increase of TNF-α and IL-1ß. In contrast, HGFs incubated with P. gingivalis exposed to cystatin C showed a decreased production of both cytokines, whereas IL-10 was enhanced. Incubation of HGFs with P. gingivalis led to an increase of nitric oxide (NO) and ROS production, which was reduced in the presence of the peptide. Conclusions: Cystatin C inhibits the growth of P. gingivalis and decreases the inflammatory cytokines, ROS, and NO production during infection of HGFs with P. gingivalis. Knowledge on the antimicrobial and immunomodulatory properties of cystatin C could aid in the design of new therapeutic approaches to facilitate the elimination of this bacterium to improve the treatment of periodontal disease.

Chagas Disease in Pregnant Women from Endemic Regions Attending the Hospital General de Mexico, Mexico City.

Trypanosoma cruzi infection leads to Chagas disease (CD), a neglected tropical infection of significant public health importance in South and Central America and other, non-endemic, countries. Pregnant women and their children are of particular importance to screen as T. cruzi can be transmitted vertically. The objective of this study was to screen for T. cruzi infection among pregnant women from endemic areas seen at the Hospital General de Mexico for prenatal care, so that they and their children may be quickly connected to CD treatment. Pregnant women were recruited through the hospital prenatal clinic and screened for T. cruzi infection using a series of serological and molecular tests. Of 150 screened patients, mean age 26.8 (SD 6.4), 30 (20.0%) were positive by at least one diagnostic test. Of these, only nine (6%) were positive as determined by PCR. Diagnosis of chronic CD is difficult in endemic places like Mexico due to the limitations of current commercially available diagnostic tests. Further evaluation of diagnostic performance of various assays could improve current CD diagnostic algorithms and proper care management in these regions. Genetic variability in the parasite may also play a role in the differing assay performances seen in this study, and this may be a valuable avenue of further research.

Leishmania mexicana: Novel Insights of Immune Modulation through Amastigote Exosomes.

Exosomes are extracellular microvesicles of endosomal origin (multivesicular bodies, MVBs) constitutively released by eukaryotic cells by fusion of MVBs to the plasma membrane. The exosomes from Leishmania parasites contain an array of parasite molecules such as virulence factors and survival messengers, capable of modulating the host immune response and thereby favoring the infection of the host. We here show that exosomes of L. mexicana amastigotes (aExo) contain the virulence proteins gp63 and PP2C. The incubation of aExo with bone marrow-derived macrophages (BMMs) infected with L. mexicana led to their internalization and were found to colocalize with the cellular tetraspanin CD63. Furthermore, aExo inhibited nitric oxide production of infected BMMs, permitting enhanced intracellular parasite survival. Expressions of antigen-presenting (major histocompatibility complex class I, MHC-I, and CD1d) and costimulatory (CD86 and PD-L1) molecules were modulated in a dose-dependent fashion. Whereas MHC-I, CD86 and PD-L1 expressions were diminished by exosomes, CD1d was enhanced. We conclude that aExo of L. mexicana are capable of decreasing microbicidal mechanisms of infected macrophages by inhibiting nitric oxide production, thereby enabling parasite survival. They also hamper the cellular immune response by diminishing MHC-I and CD86 on an important antigen-presenting cell, which potentially interferes with CD8 T cell activation. The enhanced CD1d expression in combination with reduction of PD-L1 on BMMs point to a potential shift of the activation route towards lipid presentations, yet the effectivity of this immune activation is not evident, since in the absence of costimulatory molecules, cellular anergy and tolerance would be expected.

TNF contributes to T-cell exhaustion in chronic L. mexicana infections of mice through PD-L1 up-regulation.

Leishmania mexicana can produce chronic infections leading to exhausted T cell phenotypes, mediated by PD-1/PD-L1. Little is known on mechanisms that induce these inhibitory molecules in chronic leishmaniasis. We analyzed factors that contribute to exhausted phenotypes in chronic L. mexicana infections of mice. Our results show that draining lymph node cells express enhanced levels of PD-1/PD-L1. T lymphocytes producing low cytokine levels were also found. L. mexicana infection of dendritic cells (DCs) produced elevated amounts of TNF and showed up-regulation of PD-L1 expression. We provide evidence that T cells of chronic L. mexicana infections in mice are functionally exhausted due to chronic TNF production, which leads to PD-L1 up-regulation in DCs. We conclude that TNF has a fundamental role in promoting T cell exhaustion during chronic L. mexicana infections, which contributes to the inability of T cells to proliferate and produce pro-inflammatory cytokines, thus favoring disease progression.

Lyme disease and relapsing fever in Mexico: An overview of human and wildlife infections.

Lyme borreliosis and Relapsing fever are considered emerging and re-emerging diseases that cause major public health problems in endemic countries. Epidemiology and geographical distribution of these diseases are documented in the US and in Europe, yet in Mexico, studies are scarce and scattered. The aims of this study were (1) to present the first confirmatory evidence of an endemic case of Lyme disease in Mexico and (2) to analyze the epidemiological trend of these both diseases by compiling all the information published on Borrelia in Mexico. Two databases were compiled, one of human cases and another of wild and domestic animals in the country. The analysis included the evaluation of risk factors for the human population, the diversity of Borrelia species and their geographic distribution. Six Borrelia species were reported in a total of 1,347 reports, of which 398 were of humans. Women and children from rural communities were shown to be more susceptible for both Lyme borreliosis and Relapsing fever. The remaining reports were made in diverse mammalian species and ticks. A total of 17 mammalian species and 14 tick species were recorded as hosts for this bacterial genus. It is noteworthy that records of Borrelia were only made in 18 of the 32 states, mainly in northern and central Mexico. These results highlight the importance of performing further studies in areas where animal cases have been reported, yet no human studies have been done, in order to complete the epidemiological panorama for Lyme borreliosis and Relapsing fever. Finally, the search for Borrelia infections in other vertebrates, such as reptiles and amphibians is recommended to gain a more accurate view of Borrelia species and their distribution. The geographical approach presented herein justifies an intense sampling effort to improve epidemiological knowledge of these diseases to aid vector control and prevention programs.

Relevance of epidemiological surveillance in travelers: an imported case of Leishmania tropica in Mexico.

We report the case of a patient with cutaneous leishmaniasis who showed a rapidly progressing ulcerative lesion after traveling to multiple countries where different Leishmania species are endemic. Diagnosis of Leishmania tropica, an exotic species in Mexico was established by using serological and molecular tools.

Simultaneous Detection and Photocatalysis Performed on a 3D Graphene/ZnO Hybrid Platform.

The synergy between graphene foam (Gf) and ZnO nanoparticles (NPs) allows the detection of analytes at low conentrations, which can be subsequently photocatalyzed on the hybrid surface as well as in the liquid phase upon illumination with low-power UV-vis light-emitting diode (LED) lamps. Detection of methylene blue (MB) and bisphenol A (BPA) is monitored either by graphene-enhanced Raman scattering (GERS) or molecular doping/sensing upon analyte adsorption. Using GERS, we were able to detect concentrations as low as 0.3 ppm of MB, which remained adsorbed on the graphene surface after a photocatalytic conversion of 88% (total conversion). The photocatalysis performances of BPA and MB performed in the liquid phase were lower and corresponded to 73 and 33% as indicated by gas chromatography-mass spectrometry (GC/MS) and UV-vis, respectively. The kinetics of photocatalysis was fitted with a quasi-first-order reaction, and the apparent rate constant (kapp) was calculated according to the Langmuir-Hinshelwood model. The fastest kinetics was achieved with the hybrid platform named "Gf-ZnO400", which was thermally treated at high temperatures and with most of the Ni etched away. This is consistent with the excellent electronic interaction between ZnO and graphene foam as indicated by photoelectrochemistry analysis. We mainly employed Raman scattering and UV-vis spectroscopy analyses for detection and photocatalysis applications; however, we also used other complementary techniques such as focused ion-beam scanning electron microscopy (FIB-SEM), X-ray photoelectron spectroscopy (XPS), diffuse reflectance, GC/MS, and photoelectrochemistry to explore the synergetic behavior of these two nanomaterials. This work brings about new insights into the detection of analyte molecules followed by photocatalysis performed in the solid and liquid states.

The mKate fluorescent protein expressed by Leishmania mexicana modifies the parasite immunopathogenicity in BALB/c mice.

Parasites have been engineered to express fluorescent reporter proteins, yet the impact of red fluorescent proteins on Leishmania infections remains largely unknown. We analysed the infection outcome of Leishmania mexicana parasites engineered for the constitutive expression of mKate protein and evaluated their immunogenicity in BALB/c mice. Infection of BALB/c mice with mKate transfected L. mexicana (LmexmKate ) parasites caused enlarged lesion sizes, leading to ulceration, and containing more parasites, as compared to LmexWT . The mKate protein showed immunogenic properties inducing antibody production against the mKate protein, as well as enhancing antibody production against the parasite. The augmented lesion sizes and ulcers, together with the more elevated antibody production, were related to an enhanced number of TNF-α and IL-1ß producing cells in the infected tissues. We conclude that mKate red fluorescent protein is an immunogenic protein, capable of modifying disease evolution of L. mexicana.

NKT cell activation by Leishmania mexicana LPG: Description of a novel pathway.

NKT cells have been associated with protection against Leishmania donovani, yet their role in infections with Leishmania mexicana has not been addressed, nor has the activation pathway been defined after stimulation with Leishmania mexicana lipophosphoglycan (LPG). We analyzed the activation of NKT cells and their cytokine production in response to Leishmania mexicana LPG. Additionally we compared NKT-cell numbers and cytokine profile in lymph nodes of skin lesions induced by Leishmania mexicana in BALB/c and C57BL/6 mice. We show that LPG activates NKT cells primarily through the indirect pathway, initiating with TLR2 stimulation of dendritic cells (DC), thereby enhancing TLR2, MHC II, and CD86 expressions and IL-12p70 production. This leads to IFN-γ production by NKT cells. C57BL/6 mice showed enhanced DC activation, which correlated with augmented IFN-γ production by NKT cells. Additionally, infected C57BL/6 mice showed elevated percentages of NKT cells with higher IFN-γ and IL-4 production in lymph nodes. We conclude that the response of NKT cells towards Leishmania mexicana LPG initiates with the indirect activation, after binding of LPG to TLR2 in DC. This indirect activation pathway enables NKT cells to produce IFN-γ during the innate phase of Leishmania infection, the magnitude of which differs between mouse strains.

Canine leishmaniasis in México: the detection of a new focus of canine leishmaniasis in the state of Guerrero correlates with an increase of human cases

Background. In Mexico, a steady increase of patients with visceral leishmaniasis has been reported, especially in the states of Chiapas and Guerrero, yet only limited information exists on canine leishmaniasis in areas of visceral leishmaniasis in Mexico. A veterinary report of dogs with nonhealing cutaneous lesions in Pungarabato, Guerrero led us to investigate the possible presence of Leishmania infection in an area where Lutzomyia longipalpis and Lutzomyia evansi, both vectors of Leishmania infantum, have been described. Methods. We analyzed skin lesions of 25 dogs by immunohistochemistry and PCR. Results. We found a 60% prevalence of Leishmania-infected dogs, the infection rate being higher in males than females. Thus, we established a new focus of canine leishmaniasis, and although to date no patients have been reported in this municipality, it is close to and shares the same ecological characteristics of dry tropical forests as regions where visceral leishmaniasis has been reported in Mexico. We also include updated information of localities of visceral leishmaniasis in Mexico as well as the distribution of possible sand fly vectors. Conclusions. Our data show the need to ascertain the magnitude of this new focus in view of the current data on human visceral leishmaniasis, a disease that is surging in Mexico.

CD8 cells of patients with diffuse cutaneous leishmaniasis display functional exhaustion: the latter is reversed, in vitro, by TLR2 agonists.

Leishmania mexicana (Lm) causes localized (LCL) and diffuse (DCL) cutaneous leishmaniasis. DCL patients have a poor cellular immune response leading to chronicity. It has been proposed that CD8 T lymphocytes (CD8) play a crucial role in infection clearance, although the role of CD8 cytotoxicity in disease control has not been elucidated. Lesions of DCL patients have been shown to harbor low numbers of CD8, as compared to patients with LCL, and leishmanicidal treatment restores CD8 numbers. The marked response of CD8 towards Leishmania parasites led us to analyze possible functional differences between CD8 from patients with LCL and DCL. We compared IFNγ production, antigen-specific proliferation, and cytotoxicity of CD8 purified from PBMC against autologous macrophages (MO) infected with Leishmania mexicana (MOi). Additionally, we analyzed tissue biopsies from both groups of patients for evidence of cytotoxicity associated with apoptotic cells in the lesions. We found that CD8 cell of DCL patients exhibited low cytotoxicity, low antigen-specific proliferation and low IFNγ production when stimulated with MOi, as compared to LCL patients. Additionally, DCL patients had significantly less TUNEL+ cells in their lesions. These characteristics are similar to cellular "exhaustion" described in chronic infections. We intended to restore the functional capacity of CD8 cells of DCL patients by preincubating them with TLR2 agonists: Lm lipophosphoglycan (LPG) or Pam3Cys. Cytotoxicity against MOi, antigen-specific proliferation and IFNγ production were restored with both stimuli, whereas PD-1 (a molecule associated with cellular exhaustion) expression, was reduced. Our work suggests that CD8 response is associated with control of Lm infection in LCL patients and that chronic infection in DCL patients leads to a state of CD8 functional exhaustion, which could facilitate disease spread. This is the first report that shows the presence of functionally exhausted CD8 T lymphocytes in DCL patients and, additionally, that pre-stimulation with TLR2 ligands can restore the effector mechanisms of CD8 T lymphocytes from DCL patients against Leishmania mexicana-infected macrophages.

Leishmania mexicana lipophosphoglycan differentially regulates PKCalpha-induced oxidative burst in macrophages of BALB/c and C57BL/6 mice.

Leishmania are protozoan parasites that infect macrophages and their survival is partially achieved through inhibition of the cellular oxidative burst by parasite lipophosphoglycan (LPG). PKCalpha is the predominant PKC isoenzyme required for macrophage oxidative burst, yet it is not known if different susceptibility of BALB/c and C57BL/6 mice to Leishmania mexicana could be related to PKCalpha. We analysed the effect of L. mexicana promastigotes and parasite LPG on expression of PKCalpha and on its activity in macrophages of both mouse strains. Our data show that expression of the isoenzyme was not altered either by LPG or by L. mexicana promastigotes. Yet LPG exerted opposing effects on PKCalpha activity of macrophages between both strains: in susceptible BALB/c cells, it inhibited PKCalpha activity, whereas in the more resistant strain it augmented enzymatic activity 2.8 times. In addition, LPG inhibited oxidative burst only in susceptible BALB/c macrophages and the degree of inhibition correlated with parasite survival. Promastigotes also inhibited PKCalpha activity and oxidative burst in macrophages of BALB/c mice, whereas in C57BL/6, they enhanced PKCalpha activity and oxidative burst inhibition was less severe. Our data indicate that control of PKCalpha-induced oxidative burst by L. mexicana LPG relates with its success to infect murine macrophages.

The efficacy of pentamidine combined with allopurinol and immunotherapy for the treatment of patients with diffuse cutaneous leishmaniasis.

A new treatment regimen was tested on patients with incurable diffuse cutaneous leshmaniasis (DCL) infected with Leishmania mexicana mexicana in Mexico. Two patients with advanced stages of the disease were treated with polychemotherapy (pentamidine and allopurinol) combined with recombinant human interferon-gamma (rIFN-gamma). For determination of the best medication, parasites isolated from patient lesions were exposed to available drugs both as promastigotes and as intracellular amastigotes. A synergistic effect was observed in vitro for the combination of pentamidine and allopurinol. Both patients were treated and recovered rapidly, but one of them developed insulin-dependent type I diabetes because of pentamidine toxicity. The complication was controlled and both patients were discharged with an apparent parasitologic cure, but after 3 months the two patients began to relapse. Our results suggest that allopurinol-pentamidine polychemotherapy, involving reduced dosage of pentamidine, combined with rIFN-gamma is an alternative for DCL patients infected with L. m. mexicana.